ES2425115T3 - Screening method for the hypersensitivity reaction to a drug - Google Patents

Abstract

A method of screening a human subject as an aid to predicting response to abacavir administration, comprising determining whether the subject has an "A" allele at the G (-237) A polymorphic site of TNFα (SEQ ID NO: 1), in which the presence of said TNFα genotype indicates that the subject has a higher risk of a hypersensitivity reaction to abacavir, compared to the risk of individuals without that allele.

Description

Screening method for the hypersensitivity reaction to a drug

Background

Hypersensitivity reactions (HSR) are unexpected immune-type reactions (allergy) that appear in a minority of patients treated with antiretroviral therapy. No symptoms or laboratory tests that predict or diagnose these events have been discovered. The usual symptoms, which appear in combinations, include fever, rashes, gastrointestinal reactions, severe fatigue and respiratory symptoms. These hypersensitivity reactions constitute a distinct clinical entity and are not the simple rashes (mild rashes without systemic symptoms) that are common reactions to many drugs. Hypersensitivity reactions are resolved when the causative drug is stopped, but they return when the intake is restarted. The exact mechanism of hypersensitivity reactions is unknown.

Antiretroviral therapy has proven effective for the treatment of individuals infected with human immunodeficiency virus (HIV) or diagnosed with acquired immunodeficiency syndrome (AIDS). Therapy with combinations of antiretroviral agents may prolong survival and decrease the risk of complications of HIV-1 infection. Adverse reactions can occur with any antiretroviral agent, some of which have the potential to cause severe morbidity and mortality (see, for example, Samuel et al., Antiretroviral Therapy, 2000, Arch. Pharm. Res., 23: 425 (2000 ); Carr et al., Lancet, 356: 1423 (2000)). Common, and usually less severe, adverse reactions include nausea, headache, fatigue, diarrhea, and non-severe skin rashes. Less common, but sometimes serious, adverse reactions to antiretroviral agents include severe skin rashes, pancreatitis, lactic acidosis, and hypersensitivity reactions.

Abacavir (Ziagen) hypersensitivity reactions have been reported in approximately 5% of patients receiving this agent alone or in combination with other antiretroviral agents (note that Ziagen is indicated for the treatment of HIV-1 infection in combination with other antiretroviral agents). The cessation of taking abacavir leads to the resolution of the symptoms of the hypersensitivity reaction. The continued administration of abacavir before an ongoing reaction or the reintroduction of abacavir in patients with a previous history of reaction may produce a sudden, serious and life-threatening reaction.

A screening test to identify subjects who are at a higher risk of presenting a hypersensitivity reaction to a pharmaceutical compound would be useful in clinical medicine.

Summary of the invention

A first aspect of the present invention is a method for identifying genotypes that confer a greater or lesser risk of a hypersensitivity reaction to abacavir in human subjects. In a population of test subjects, each subject is genotyped to detect polymorphisms in a candidate gene, such as the TNFalfa (TNFα) gene, MICA, MICB and / or the HLA genes. A therapeutic regimen of abacavir is administered to each subject (before, at the same time or after genotyping of the subject), and the test subjects that show (or show) clinical signs of a hypersensitivity reaction to abacavir are identified. The genotypes of the test subjects at the polymorphic sites in the candidate genes correlate with the appearance of clinical signs of hypersensitivity reaction, to determine which genotypes are associated with a greater or lesser risk of a hypersensitivity reaction (compared to others genotypes or with a general population that has not been stratified according to genotype).

Another aspect of the present invention is a method of determining if an individual has a higher risk of suffering a hypersensitivity reaction to abacavir, determining whether the individual has a genotype that is associated with a higher risk of a hypersensitivity reaction, compared to the risk in subjects with other genotypes.

Another aspect of the present invention is a method of determining if an individual has a lower risk of suffering a hypersensitivity reaction to abacavir, determining whether the individual has a genotype that is associated with a lower risk of a hypersensitivity reaction, compared to the risk in subjects with other genotypes.

Another aspect of the present invention is a method of screening a human subject as an aid to assess the suitability of abacavir administration, determining whether the subject has a genotype of TNFα that has been associated with an increased risk of a hypersensitivity reaction against on abacavir, compared to the risk in subjects with other TNFα genotypes. The presence of said TNFα genotype indicates that the subject has a higher risk of a hypersensitivity reaction to abacavir.

Another aspect of the present invention is a method of screening a human subject as an aid to assess the suitability of abacavir administration, determining whether the subject has an HLA genotype that has been associated with an increased risk of a hypersensitivity reaction against abacavir, compared to the risk in subjects with other HLA genotypes. The presence of said HLA genotype indicates that the subject has a higher risk of a hypersensitivity reaction to abacavir.

A method for treating a human subject with abacavir is also described, first genotyping the subject to detect the presence or absence of the HLA-B57 allele, and then administering abacavir if the HLA-B57 allele is not detected.

Another aspect of the present invention is a method of screening a human subject, as an aid to predict the subject's risk of undergoing a hypersensitivity reaction to a therapeutic regimen of abacavir, genotyping a DNA sample of the subject to determine the presence of a polymorphism in the TNFα gene, in which polymorphism has previously been associated with an increased risk of HSR by abacavir, compared to the risk of HSR associated with other TNFα polymorphisms. Detection of the presence of a TNFα genotype that has been associated with a higher incidence of abacavir hypersensitivity reactions (compared to the incidence of abacavir HSR associated with other TNFα genotypes) indicates that the subject has a higher risk of a abacavir hypersensitivity reaction.

Another aspect of the present invention is a method of screening a human subject, as an aid to predict the subject's risk of undergoing a hypersensitivity reaction to a therapeutic regimen of abacavir, genotyping a DNA sample of the subject to determine the presence of a polymorphism in an HLA gene, in which polymorphism has previously been associated with an increased risk of HSR by abacavir, compared to the risk of HSR associated with other polymorphisms. The presence of an HLA genotype that has been associated with a higher incidence of abacavir hypersensitivity reactions (compared to the incidence of abacavir HSR associated with other HLA genotypes) indicates that the subject has a higher risk of a hypersensitivity reaction. Abacavir

A method to identify human genotypes associated with an increased risk of a hypersensitivity reaction to abacavir is also described, by genotyping each member of a population of test subjects for at least one polymorphism in the TNFα gene, the administration of a regimen therapeutic abacavir to each test subject, and the identification of the test subjects showing clinical signs of a hypersensitivity reaction to abacavir. The correlation of TNFα genotypes with the appearance of clinical signs of a hypersensitivity reaction will determine which genotypes are associated with an increased risk of abacavir hypersensitivity reaction (compared to the other genotypes detected).

A method to identify human genotypes associated with an increased risk of a hypersensitivity reaction to abacavir is also described, by genotyping each member of a population of test subjects for at least one polymorphism in the HLA gene, the administration of a regimen therapeutic abacavir to each test subject, and the identification of the test subjects showing clinical signs of a hypersensitivity reaction to abacavir. The correlation of HLA genotypes with the appearance of clinical signs of a hypersensitivity reaction will determine which genotypes are associated with an increased risk of abacavir hypersensitivity reaction (compared to the other genotypes detected).

Another aspect of the present description is a method of administering or prescribing abacavir to reduce the occurrence of a hypersensitivity reaction to abacavir. The method comprises selecting, based on the genotype status, a treatment population from a population of major initial subjects who have a disorder suitable for abacavir treatment. The treatment population is selected to increase the percentage of subjects in the treatment population that have a genotype that has been associated with a lower risk of a hypersensitivity reaction to abacavir (the highest percentage of subjects in the treatment population is in relation to to the percentage of subjects in the initial population). Alternatively, the treatment population is selected to decrease the percentage of subjects in the treatment population that have a genotype that has been associated with an increased risk of abacavir hypersensitivity reaction. Then abacavir is administered to the selected treatment population, thereby reducing the incidence of HSR by abacavir in the treated population, compared to the incidence that would have been expected to occur if abacavir had been administered in the older initial population. "Screening" can be produced by any suitable process, as will be apparent to those skilled in the art. Examples of suitable screening methods include the genetic screening of individuals in the initial population, or otherwise classifying subjects by genotype (for example, when a subject's genotype is known, it is not necessary to repeat the genetic test) ; or otherwise regulate access to abacavir to decrease the number of subjects in the treatment population that have genotypes that have been associated with an increased risk of HSR by abacavir. One of these genotypes is the HLA-B57 allele, with which the treatment population will be selected to minimize the appearance of the HLA-B57 allele in the treatment population. Alternatively, the genotype of interest may be the TNF G (-237) A polymorphism, whereby the treatment population will be selected to minimize the appearance of the A allele.

Detailed analysis

Antiretroviral therapy in HIV-infected patients often includes the use of multiple types of antiretroviral agents, including protease inhibitors, non-nucleoside reverse transcriptase inhibitors (NNRTIs) and nucleoside reverse transcriptase inhibitors (NRTIs). Abacavir is a purine nucleoside analog that is commercially available as abacavir sulfate (ZIAGEN®, GlaxoSmithKline), and is used in combination with other antiretroviral agents to treat HIV-infected subjects. Abacavir is an HIV-1 reverse transcriptase inhibitor (NRTI) that contains an unsaturated cyclopentene ring instead of the 2'deoxyriboside of natural deoxynucleosides, and contains a cyclopropylamino group. The chemical name of abacavir sulfate is (cis) -4- [2-amino-6- (cyclopropylamino) -9H-purin-9-yl] -2-cyclopenten-1-methanol (salt) sulfate (2: 1 ).

Hypersensitivity reactions are idiosyncratic events of a supposed immunological nature that appear with a wide range of pharmacological compounds. In the context of the administration of abacavir, hypersensitivity reactions to abacavir can be serious and progress to life-threatening (Clay et al., Ann. Pharmacotherapy, 34 (2): 247 (2000); Staszewski et al. , AIDS, 12: F197 (1998)). In clinical trials, abacavir hypersensitivity has occurred in approximately 5% of subjects. Signs and symptoms of a hypersensitivity reaction to abacavir include, but are not limited to fever, skin rashes, fatigue, gastrointestinal symptoms (including nausea, vomiting, diarrhea, abdominal pain), and respiratory symptoms (including pharyngitis , dyspnea and cough). Other signs and symptoms include malaise, myalgia, myolysis, arthralgia, edema, headache and paraesthesia. Physical results include lymphadenopathy, lesions in mucous membranes (conjunctivitis and canker sores). The rash associated with the hypersensitivity reaction usually has a maculopapular or urticarial appearance, but the appearance may be variable; Up to 30% of hypersensitivity reactions have appeared without a rash. Laboratory abnormalities include liver function tests with high results, increased creatine phosphokinase or creatinine, and lymphopenia. See the insert in the package of Ziagen (abacavir sulfate), Glaxo Wellcome, Research Triangle Park, NC (1998); Clay et al., Management Protocol for Abacavir-related Hypersensitivity Reaction, Ann. Pharmacotherapy, 34 (2): 247 (2000). Clay et al. indicate that only the presence of a rash does not justify the abandonment of abacavir, unless other systemic symptoms of a hypersensitivity reaction appear.

TNFalfa

It is known that the immunological effector molecule tumor necrosis factor-alpha (TNFα) is polymorphic, and a series of polymorphisms have been indicated in the TNFα promoter region. Some reports indicate that such promoter polymorphisms influence immune disease (Bouma et al., Scand. J. Immunol., 43: 456 (1996); Allen et al., Mol. Immunology, 36: 1017 (1999)), while others suggest that the associations observed between TNFα polymorphisms and disease occurrence are not due to the functional effects of TNFα, but to the linkage imbalance of TNFα with selectable HLA alleles (Uglialoro et al., Tissue Antigens, 52: 359 (1998)). A list of TNFα promoter polymorphisms is provided by Allen et al., Mol. Immunology, 36: 1017 (1999). The numbering of TNFα polymorphisms varies among authors, due to the variation in the sequences indicated for the TNFα promoter region; herein, the numbering refers to the following consensus sequence provided in Allen et al. (1999):

The transcription start site (+1) is indicated by bold and underlined; polymorphisms G (-237) A and G (308) A are indicated with bold and double underlined. Due to the variation in the sequences and numbering indicated, polymorphism G (-237) A has also been referred to as G-238A, and polymorphism G (-308) A is located at position -307 of the previous sequence. Another polymorphism, C (-5,100) G, investigated in the present investigation, is a C / G polymorphism in the 5 'untranslated region of TNFα:

N = C / G

Allen et al. (supra) warn that the number of TNFα promoter polymorphisms observed to date are

10 G / A polymorphisms grouped in the region from -375 to -162 pp; that some of these polymorphisms are within a common motive; and suggest that the motive may be a consensus binding site for a transcriptional regulator or that it may influence the structure of the DNA. It has been indicated that G / A polymorphism in -237 affects the curvature of DNA (D'Alfonso et al., Immunogenetics, 39: 150 (1994)). Huizinga et al. (J. Neuroimmunology, 72: 149, 1997) indicate a significantly lower production of TNFα in stimulated cells

15 with LPS in heterozygous individuals (G / A) in -237 (compared to G / G individuals); however, another study did not observe these effects (Pociot et al., Scand. J. Immunol., 42: 501, 1995). It has also been indicated that polymorphism G (-237) A affects autoimmune diseases (Brinkman et al., Br. J. Rheumatol., 36: 516, 1997 (rheumatoid arthritis); Huizinga et al., J. Neuroimmunology, 72 : 149, 1997 (multiple sclerosis); Vinasco et al., Tissue Antigens, 49:74, 1997 (rheumatoid arthritis)) and infectious diseases (Hohler et al., Clin. Exp. Immunol., 111: 579,

20 1998 (hepatitis B); Hohler et al., J. Med. Virol., 54: 173, 1998 (hepatitis C)).

It is known that the genetic, nucleotide and amino acid sequences obtained from different sources for the same gene can vary both in the numbering scheme and in the precise sequence. These differences may be due to the sequence variability inherent within the gene and / or to sequencing errors. Accordingly, those skilled in the art will understand that the reference herein to a particular polymorphic site

25 with a number (for example, TNFα G-238A) includes polymorphic sites that correspond in sequence and location within the gene, even if different numbering / nomenclature schemes are used to describe them.

HLA

The HLA complex of humans (major histocompatibility complex or MHC) is a cluster of genes

30 related located on chromosome 6 (the TNFα and HLA B loci are very close on chromosome 6). The HLA complex is classified into three regions: Class I, II, and III regions (Klein J., in: Gotze D, ed., The Major Histocompatibility System in Man and Animals, New York, Springer-Verlag, 1976: 339-378). Class I HLAs comprise the transmembrane protein (heavy chain) and a beta-2 microglobulin molecule. Class I transmembrane proteins are encoded by the HLA-A, HLA-B and HLA-C loci. The function of HLA molecules

Class I is to present antigenic peptides (including viral protein antigens) to T cells. Three isoforms of MHC class II molecules, designated HLA-DR, -DQ, and -DP, are recognized. MHC class II molecules are heterodimers composed of an alpha chain and a beta chain; Different alpha and beta chains are encoded by subsets of A genes and B genes, respectively. Various HLA-DR haplotypes have been recognized, and they differ in the organization and number of DRB genes present on each DR haplotype; they have

40 described multiple DRB genes (Bodmer et al., Eur. J. Immunogenetics, 24: 105 (1997); Andersson, Frontiers in Bioscience, 3: 739 (1998)).

The MHC shows a high polymorphism; more than 200 genotypic alleles of HLA-B have been indicated. See, for example, Schreuder et al., Human Immunology, 60: 1157-1181 (1999); Bodmer et al., European Journal of Immunogenetics, 26: 81-116 (1999). Despite the number of alleles in the HLA-A, HLA-B and HLA-C loci, the number of haplotypes observed in populations is less than what would be expected mathematically. Certain alleles tend to appear together on the same haplotype, rather than randomly segregated. This is called linkage disequilibrium (LD) and can be quantified by methods known in the art (see, for example, Devlin and Risch, Genomics, 29: 311 (1995); BS Weir, Genetic Data Analysis II , Sinauer Associates, Sunderland, MD (1996)).

Products encoded by polymorphic HLA loci are typically typified by serological methods for the histocompatibility test of transplants and transfusions, and blood component therapy. Serological typing is based on reactions between characterized sera and HLA gene products. Known techniques for histocompatibility testing include microlinphocytotoxicity and flow cytometry. The usual microlinphocytotoxicity for HLA antigen typing determines the HLA antigen profile of a subject's lymphocytes, using a panel of well characterized HLA antisera. The HLA-B57 allele is well characterized, and serological methods for detecting HLA-B57 are known. See, for example, ASHI Laboratory Manual, 4th edition, American Society for Histocompatibility and Immunogenetics (2000); Hurley et al., Tissue Antigens, 50: 401 (1997).

More recently, methods for the analysis of HLA polymorphisms at the genetic level have been developed. Non-serological HLA typing methods include the use of DNA restriction fragment length polymorphism (RFLP; see, for example, Erlich, US Patent No. 4,582,788 (1986)), or labeled oligonucleotides, for Identify specific HLA DNA sequences. These methods can detect polymorphisms located in the coding or non-coding sequence of the genome. See, for example, Bidwell et al., Immunology Today, 9:18 (1988); Angelini et al., Proc. Natl Acad. Sci. USA, 83: 4489 (1986); Scharf et al., Science, 233: 1076 (1986); Cox et al., Am. J. Hum. Gen., 43: 954 (1988); Tiercy et al., Proc. Natl Acad. Sci. USA, 85: 198 (1988); and Tiercy et al., Hum. Immunol., 24: 1 (1989). The polymerase chain reaction (PCR) process (see US Patent No. 4,683,202, 1987) allows the amplification of genomic DNA and is now used for HLA typing procedures. See, Saiki et al., Nature, 324: 163 (1986); Bugawan et al., J. Immunol., 141: 4024 (1988); Gyllensten et al., Proc. Natl Acad. Sci. USA, 85: 7652 (1988). See also, for example, Ennis et al., PNAS USA, 87: 2833 (1990); Petersdorf et al., Tissue Antigens, 46:77 (1995); Girdlestone et al., Nucleic Acids Research, 18: 6702 (1990); Marcos et al., Tissue Antigens, 50: 665 (1997); Steiner et al., Tissue Antigens, 57: 481 (2001); Madrigal et al., J. Immunology, 149: 3411 (1992).

As used herein, "genotyping" an HLA locus refers to methods that identify the presence or absence of a particular allele, or of a nucleic acid or amino acid sequence; sequence variations can be detected directly (by sequencing) or indirectly (for example, by analysis of fragment length polymorphisms, or detection of hybridization of a probe of known sequence, or reference strand conformation polymorphism). HLA alleles can be detected serologically, as is known in the art.

Differentiated HLA alleles have been associated with a greater or lesser risk of progression of HIV disease. HLA-B57 and HLA-B14 alleles have been associated with non-progressive HIV infection, while HLA-A29 and HLA-B22 have been associated with rapid progression (Goulder et al., J. Virology, 74: 5291 ( 2000); Hendel et al., J. Immunology, 162: 6942 (1999)). Carrington et al. have indicated that the frequency of the HLA-B57 allele in cohorts of HIV-infected patients is 4.40% in Caucasians and 5.7% in African Americans (Carrington et al., Science, 283: 1748 (1999)).

MICA and MICB

Gene A related to the MHC class I (HLA) chain (MICA) and gene B related to the MHC class I (HLA) chain (MICB) belong to a family of multi-copy genes located in the region of the major histocompatibility complex (MHC) of class I near the HLA-B gene. They are located within a binding region on chromosome 6p around HLA-B and TNFalfa. MHC class I encoded molecules are induced by stress factors, such as infection and thermal shock, and are expressed on the gastrointestinal epithelium.

It has been indicated that MICA is highly polymorphic. The occurrence of polymorphisms of a single nucleotide of MICA in various ethnic groups has been indicated by Powell et al., Mutation Research, 432: 47 (2001). It has been indicated that polymorphisms in MICA are associated with various diseases, although in some cases the association has been attributed to a linkage imbalance with the HLA genes. See, for example, Salvarani et al., J. Rheumatol., 28: 1867 (2001); Gonzalez et al., Hum. Immunol., 62: 632 (2001); Seki et al., Tissue Antigens, 58:71 (2001).

Various polymorphic forms of MICB have been indicated (see, for example, Visser et al., Tissue Antigens, 51: 649 (1998); Kimura et al., Hum. Immunol., 59: 500 (1998); Ando et al. , Immunogenetics, 46: 499 (1997); Fischer et al., Eur.

J. Immunogenet., 26: 399 (1999)).

Next, a partial sequence is provided for the Homo sapiens MICA gene, which includes exons 2 and 3 (GenBank, reference AJ295250).

In the present study, various MICA polymorphisms have been investigated. MICA polymorphisms in exon 2 (T / G; rs1063630 in the National Center for Biotechnology Information SNP database (dbSNP)) and exon 3 (A / G; rs1051792) are shown in bold and double underlined above . Another MICA polymorphism investigated in the present study (rs1052416) is located approximately -9,263 5 'bases to the transcription initiation site:

MICA (-9,263)

10 A complete cds for the human MICB gene is provided in SEQ ID NO: 4 (GenBank, register U65416). The MICB polymorphisms investigated in the present study include one in exon 2 (rs1065075) and another in exon 3 (rs1051788):

MICB - (rs1065075) N = A / G GTGGGCAGAAGATGTCCTGGGAGCTNAGACCTGGGACACAGAGACCGAGGA, SEQ ID NO: 5

15 MICB (rs1051788) N = A / G CAGGGGCTCCCGGCATTTCTACTACNATGGGGAGCTCTTCCTCTCCCAAAA, SEQ ID NO: 6

P47-dependent ATP-dependent helicase RNA

The protein encoded by this gene is a member of the ATP-dependent family of RNA helicases, and is also called transcription 1 associated with HLA-B 1 (BAT1) (see, for example GenBank, registration number AF029061). 20 A cluster of genes known as BAT1-BAT5 has been located near TNF and TNF genes. Several polymorphisms have been identified in the ATP-dependent p47 RNA helicase, including:

N = A / T

RS929138; N = C / T CTTTGGCAATTCTATATGGTGAGCTNTAAAGGTGGGCTCCAGGTAGGGATG, SEQ ID NO: 8

25 Definitions

It is known that the genetic, nucleotide and amino acid sequences obtained from different sources for the same gene can vary both in the numbering scheme and in the precise sequence. These differences may be due to numbering schemes, sequence variability inherent within the gene and / or sequencing errors. Accordingly, those skilled in the art will understand that the reference herein to a particular polymorphic site with a number (eg, TNFα G-238A) includes the polymorphic sites that are

they correspond in sequence and location within the gene, even if different numbering / nomenclature schemes are used to describe them.

As used herein, a "hypersensitivity reaction" (HSR) to a drug refers to the development of an immune response to a molecule of a drug or a metabolite of the drug. This response is generally characterized by multiple symptoms and is consistent with the clinical descriptions of these syndromes (Knowles et al., Lancet, 356: 1587 (2000); Carr et al., Lancet, 356: 1423, (2000)). The immune reaction shares characteristics, but is not necessarily identical, with the types present in the Gell and Coombs system. See, Sullivan, T.J., Drug allergy, in Middleton et al. (eds.), Allergy: Principles and Practice, 4th ed., St. Louis, Mosby, 1993, p. 1730. HSR at abacavir can be characterized by the appearance of multiple symptoms or individual symptoms, and the clinical diagnosis of HSR at abacavir or probable HSR at abacavir can be established based on the presence of one or more clinical signs and symptoms, physical findings, with or without laboratory abnormalities, as will be apparent to those skilled in the art.

The administration of abacavir to a subject (or "treating" a subject with abacavir) comprises methods and routes of administration known in the art. The recommended therapeutic regimens (dosage and programming amounts, plasma concentrations) of abacavir are known in the art. As used herein, the administration of abacavir is not limited to the treatment of HIV or AIDS-related disease, but includes its medical use for other disorders that can be treated with abacavir.

As used herein, the administration of a pharmaceutical reverse transcriptase inhibitor to a subject comprises the administration of an effective amount of the pharmaceutical agent to the subject in need thereof. The dosage of a pharmaceutical agent can be determined according to methods known and accepted in pharmaceutical techniques, and can be determined by those skilled in the art. Reverse transcriptase inhibitors (NRTI and NNRTI) are known for the treatment of HIV disease and / or AIDS.

As used herein, the "HLA-B57 allele" refers to an HLA-B allele that is serologically characterized as the HLA-B57 allele, as is known in the art. It will be recognized that serologically characterized HLA-B57 alleles comprise sequence variants that can be detected at the level of the nucleic acid sequence (for example, HLA-B * 5701, HLA-B * 5702; see, for example, Schreuder et al ., Human Immunology, 60: 1157-1181 (1999)).

As used herein, "genotyping" a subject (or a DNA sample) for a polymorphic allele of a gene or genes means detecting which allelic or polymorphic forms or forms of the gene or genes are present in a subject (or a sample). As is known in the art, an individual can be heterozygous or homozygous for a particular allele. There may be more than two allelic forms, so more than three genotypes may be possible. For the purposes of the present invention, "genotyping" includes the determination of HLA alleles using suitable serological techniques, as is known in the art. As used herein, an allele can be "detected" when other possible allelic variants have been discarded; for example, when it is discovered that a specified position in a nucleic acid is not adenine (A), thymine (T) or cytosine (C), it can be concluded that guanine (G) is present in that position (that is, it is "detected" "G).

As used herein, a "genetic subset" of a population consists of members of the population who have a specific genotype. In the case of a biallelic polymorphism, a population can potentially be divided into three subsets: homozygous for allele 1 (1,1), heterozygous (1,2), and homozygous for allele 2 (2,2). A "population" of subjects can be defined using various criteria, for example, individuals who are being treated with abacavir, HIV-infected individuals, individuals with a specific ethnic background. It is known that the frequency of a particular allele may differ between populations of different ethnic backgrounds. For example, it has been indicated that the allelic frequency of HLA-B57 is approximately 4% among blacks and Caucasians (therefore, approximately 8% of this population carries at least one copy of the HLA-B57 allele), but among the Japanese, has indicated that the frequency is 0.3% (Cao et al., Human Immunology, 62: 1009 (2001)). The distribution of HLA-B57 subtypes also varies with ethnicity, being> 90% of HLA-B57-positive Caucasians for the HLA-B5701 subtype, compared with approximately 60% of African Americans (Williams et al., Human Immunology, 62: 645 (2001)).

As used herein, a subject that is "predisposed" or "has a higher risk" to a specific phenotypic response, based on genotyping, will be more likely to show this phenotype than an individual with a genotype other than the locus (or loci) target polymorphic. When the phenotypic response is based on a multiallelic polymorphism, or on the genotyping of more than one gene, the relative risk may differ among the multiple possible genotypes.

A "genetic test" (also called genetic screening), as used herein, refers to the testing of a biological sample from a subject to determine the genotype of the subject, and can be used to determine whether the genotype of the subject comprises alleles that cause, or that increase the susceptibility to a particular phenotype (or that are in linkage imbalance with an allele or alleles that cause or increase susceptibility to this phenotype).

"Linkage imbalance" refers to the tendency of specific alleles in different genomic locations to appear together more often than expected by chance. The alleles in specific loci are in complete equilibrium if the frequency of any particular set of alleles (or haplotype) is the product of their frequencies in the individual population. A measurement of the linkage imbalance that is commonly used is r:

nr2 has an approximate chi-square distribution with 1 degree of freedom for biallelic markers. It is considered that the loci that show an r such that nr2 is greater than 3.84, which corresponds to a significant stacking statistic at the 0.05 level, are in linkage imbalance (BS Weir, 1996, Genetic Data Analysis II Sinauer Associates, Sunderland, MD).

Alternatively, a standardized measurement of linkage imbalance can be defined as:

The value of D 'has a range of -1.0 to 1.0. When the statistically significant absolute D 'value for two markers is not less than 0.3, they are considered to be in linkage imbalance.

As used herein, the term "an HLA-B57 genotype" refers to a genotype that includes the HLA-B57 allele. An HLA-B57 genotype can be identified by detecting the presence of an HLA-B57 allele, or by detecting a genetic marker known to be in linkage imbalance with HLA-B57.

As used herein, the determination of a "multilocus" genotype refers to the detection, within an individual, of the alleles present in more than one locus. A subject can be genetically selected to determine the presence or absence of an HLA allele (for example, the HLA-B57 allele) and a TNFα allele (for example, in the TNFα G locus (-237) A).

As used herein, the process of detecting an allele or a polymorphism includes, but is not limited to serological and genetic methods. The allele or polymorphism detected may be functionally involved in affecting the phenotype of an individual, or it may be an allele or a polymorphism that is in linkage disequilibrium with a functional polymorphism / allele. Polymorphisms / alleles manifest in the genomic DNA of a subject, but can also be detected in RNA, cDNA or protein sequences transcribed or translated from this region, as will be apparent to those skilled in the art.

Alleles, polymorphisms or genetic markers that are "associated" with HSR to an NRTI, such as abacavir, are often overrepresented in treated subjects suffering from HSR, compared to treated subjects not suffering from HSR, or compared to the general population.

According to the present methods, subjects who are being treated with abacavir, or who are considering abacavir treatment, can be selected as an aid to predict their risk of suffering a hypersensitivity reaction to abacavir. Screening comprises obtaining a biological sample of the subject and analyzing it to determine the genotype of the TNFα and / or HLA genes, that is, to determine the presence or absence of polymorphisms in one or both genes, which are associated with an increased risk of HSR at abacavir (compared to the risk associated with other polymorphisms).

The present inventors have established that there is a correlation between the HLA genotype of an individual (in particular, class I, and more particularly HLA-B) and / or the TNFα genotype, and the risk of suffering a hypersensitivity reaction to the administration of abacavir. Accordingly, a method of assessing the relative risk of an individual from HSR to abacavir involves genotyping that individual into the TNFα gene or the HLA genes to determine whether the individual's genotype exposes them to an increased risk of HSR by abacavir. Individuals who possess a TNFα or HLA genotype that have previously been associated with a higher incidence of HSR by abacavir (compared to the incidence of HSR in subjects with other genotypes) have a higher risk of HSR.

The present screening methods comprise genotyping a subject in the HLA genes, in particular the HLA class I genes, more particularly the HLA-B gene, which includes detecting the presence or absence of the HLA-B57 allele (as defined in the present).

The present screening methods also comprise genotyping a subject in the TNFα gene, and more particularly detecting the genotype at the polymorphic site of TNFα G (-237) A (as defined herein), in which the detection of a Allele A indicates an increased risk of a hypersensitivity reaction, compared to the detection of a G / G genotype.

In view of the present description, it will be apparent to those skilled in the art how to determine other TNFα and / or HLA genotypes that are associated with an increased risk of HSR by abacavir. Various allelic forms of the TNFα and HLA genes are known, and methods for typing the TNFα and HLA genes are known in the art. As other polymorphisms are detected in human TNFα and HLA genes, the typing of such polymorphisms can be based on known methods. Therefore, a population of subjects who have received abacavir can be typed, and correlate the genotype of TNFα and / or HLA with the appearance of HSR. In an alternative method, only subjects who have suffered HSR can be genotyped and, when the prevalence of a TNFα or HLA allele in a concordant control population (not HSR) is known, determine whether the allele is overrepresented in the HSR population, which indicates that it is associated with HSR. As will be apparent to those skilled in the art, the detection of a TNFα or HLA allele can be performed by typing genetic markers known to be in linkage disequilibrium with the allele / polymorphism of TNFα or target HLA. Preferably, these markers are in substantial linkage imbalance, more preferably the markers are in complete linkage imbalance.

The present invention also provides a method for assessing the relative risk of an individual suffering from HSR at abacavir, determining the genotype of both TNFα and HLA genes, to determine whether the individual's genotype exposes them to an increased risk of HSR at abacavir. Individuals who have a combined TNFα / HLA genotype that is associated with a higher incidence of HSR by abacavir (compared to the incidence of HSR in subjects with other genotypes) have a higher risk of HSR. In particular, the present methods may comprise detecting the allelic form of the TNFα G polymorphism (-237) A and the presence or absence of the HLA-B57 allele (and / or markers in linkage disequilibrium with these).

Since there are multiple genotypes of TNFα and HLA, it will be apparent to those skilled in the art that the relative risk of HSR to abacavir may vary between multiple genotypes. For example, in a multilocus screening method in which more than two genotypes are found, it can be determined that the relative risk is higher for one genotype, lower for another, and intermediate in others. It can be considered a "higher risk" when compared to the risk in a population that has not been stratified according to the genotype (a general population), or it may be higher when compared to the expected risk in another defined genotype.

Therefore, the presence of a particular predetermined genotype that is associated with an increased risk of HSR indicates a higher probability that the individual will show the associated phenotype (HSR reaction) relative to subjects with other genotypes. The genotype will rarely be absolutely predictive, that is, when a population with a certain genotype shows a high incidence of an associated phenotype, not all individuals with that genotype will show the phenotype. Similarly, some individuals with a different genotype may show the same phenotype. However, it will be apparent to those skilled in the art that the genotyping of a subject as described herein will be an aid in predicting the risk of a subject to HSR against abacavir treatment, and thus help in making treatment decisions. . The present methods may also include administering abacavir to subjects after screening, in subjects in whom the risk of HSR is considered acceptable; The final treatment decision will be based on factors other than the genetic test (as will be apparent to those skilled in the art), which include the subject's overall health status and the expected outcome of the treatment.

It will be apparent to those skilled in the art that the present methods can also be applied when hypersensitivity reactions occur in response to synthetic nucleoside analogs other than abacavir, and in particular NRTI. In particular, these compounds include purine nucleoside analogs, purine nucleoside analogs containing an unsaturated carbon ring instead of the 2'-deoxyriboside of natural deoxynucleosides, and purine nucleoside analogs containing a cyclopentene ring unsaturated in 2'-deoxyriboside site of natural deoxynucleosides. In addition, the present methods are

applicable when HSR appears in response to NNRTI, such as efavirenz (SUSTIVA ™, Dupont Pharmaceuticals)

and nevirapine (VIRAMUNE®, Boerhinger Ingelheim / Roxane).

According to the present methods, a compound (such as an NRTI or NNRTI) can be selected for variation in the incidence of HSR among genetic subpopulations of subjects. These methods include administering the compound to a population of subjects, obtaining biological samples from the subjects (which can be performed before or after administration of the compound), genotyping the polymorphic allelic sites in the TNFα gene and / or the HLA class I genes (in particular, the HLA-B gene), and correlate the genotype of the subjects with their phenotypic response (for example, the absence of a hypersensitivity reaction versus the presence of a suspected or confirmed hypersensitivity reaction). As will be apparent to those skilled in the art, due to the severe nature of HSR, it may be necessary to stop the administration of a pharmaceutical compound when a hypersensitivity reaction is suspected due to the presence of rashes and / or other symptoms compatible with the clinical syndrome The correlation of certain genotypes with a higher proportion of HSR (whether it has been confirmed or if there is clinical suspicion of HSR), compared to the incidence of HSR in subjects with other genotypes, indicates that the incidence of HSR varies among genetic subpopulations.

In other words, the methods of the present invention can be used to determine the correlation of a polymorphic allele (such as those found in the TNFα and / or HLA alleles), with the incidence of a hypersensitivity reaction to a pharmaceutical compound , in particular an NRTI. The subjects are stratified according to the genotype and their response to the therapeutic agent is evaluated (prospectively or retrospectively) and the genotypes are compared. In this way, genotypes that are associated with a higher (or lower) rate of HSR can be identified. The higher or lower HSR rate is compared with rates among other genotypes, or with a population as a whole (that is, the incidence in a population that is not stratified by genotype).

Polymorphic alleles can be detected by determining the DNA polynucleotide sequence, or by detecting the corresponding sequence in RNA transcripts from the polymorphic gene, or when the nucleic acid polymorphism produces a change in an encoded protein, by detecting such changes in the sequence of amino acids in the encoded proteins, using any suitable technique as known in the art. The polynucleotides used for typing are generally from genomic DNA, or a polynucleotide fragment from a genomic polynucleotide sequence, such as in a bank prepared using the individual's genomic material (eg, a cDNA bank). The polymorphism can be detected with a method comprising contacting a sample of polynucleotide or protein from an individual, with a specific binding agent for the polymorphism, and determining whether the agent binds to the polynucleotide or protein, indicating the binding that polymorphism is present. The binding agent can also bind to nucleotides and flanking amino acids on one or both sides of the polymorphism, for example, at least 2, 5, 10, 15 or more nucleotides or flanking amino acids in total or on each side. In the case where the presence of the polymorphism is being determined in a polynucleotide, it can be detected in the double-stranded form, but is generally detected in the single-stranded form.

The binding agent may be a polynucleotide (single or double stranded) generally with a length of at least 10 nucleotides, for example at least 15, 20, 30, or more nucleotides. A polynucleotide agent used in the method will generally bind the polymorphism of interest, and the flanking sequence, in a sequence specific manner (for example, hybridizes according to Watson-Crick base pairing) and, thus, generally has a sequence that is totally or partially complementary to the sequence of the polymorphism and the flanking region. The binding agent may be a molecule that is structurally similar to polynucleotides comprising units (such as purine or pyrimidine analogs, peptide nucleic acids, or RNA derivatives, such as blocked nucleic acid (LNA)) capable of participating in a Watson-Crick base mating. The agent can be a protein, generally with a length of at least 10 amino acids, such as at least 20, 30, 50,

or 100 or more amino acids. The agent may be an antibody (including a fragment of said antibody that is capable of binding to the polymorphism).

In one embodiment of the present methods, a binding agent is used as a probe. The probe may be labeled or be able to be labeled indirectly. Marker detection can be used to detect the presence of the (bound) probe on the individual polynucleotide or protein. The binding of the probe to the polynucleotide or protein can be used to immobilize the probe or the polynucleotide or protein (and thus separate it from a composition or solution).

In another embodiment of the present methods, the individual polynucleotide or protein is immobilized on a solid support and then contacted with the probe. The presence of the immobilized probe on the solid support (through its binding to the polymorphism) is then detected, directly by detecting a marker on the probe, or indirectly by contacting the probe with a residue that binds to the probe. In the case of detecting a polynucleotide polymorphism, the solid support in general is made of nitrocellulose or nylon. In the case of a protein polymorphism, the method can be based on an ELISA system.

The present methods can be based on an oligonucleotide coupling assay, in which two oligonucleotide probes are employed. These probes bind adjacent areas on the polynucleotide containing the polymorphism, allowing (after binding) that the two probes are coupled to each other by an appropriate ligase enzyme. However, the two probes will only bind (in a manner that allows coupling) to a polynucleotide containing the polymorphism, and therefore, the detection of the coupled product can be used to determine the presence of the polymorphism.

In one embodiment, the probe is used in a system based on heteroduplex analysis to detect polymorphisms. In this system, when a probe binds to a polynucleotide sequence containing the polymorphism, it forms a heteroduplex at the site where the polymorphism appears (that is, it does not form a double-stranded structure). This heteroduplex structure can be detected using an enzyme that is specific to a single or double strand. Generally, the probe is an RNA probe, and the enzyme used is an RNase H that breaks the heteroduplex region, thus allowing the detection of polymorphism by detecting the products of the rupture.

The method may be based on a fluorescent chemical breakdown analysis, which is described, for example, in PCR Methods and Applications, 3: 268-271 (1994), and in Proc. Natl Acad. Sci., 85: 4397-4441 (1998).

In one embodiment, the polynucleotide agent is capable of acting as a primer for a PCR reaction only if it binds to a polynucleotide containing the polymorphism (i.e., a sequence or allele specific PCR system). Thus, only one PCR product will be produced if the polymorphism is present in the individual's polynucleotide, and the presence of the polymorphism is determined by the detection of the PCR product. Preferably, the region of the primer that is complementary to the polymorphism is in or near the final 3 'region of the primer. In one embodiment of this system, the polynucleotide and the agent will bind to the wild type sequence but do not act as a primer for a PCR reagent.

The method may be a system based on restriction fragment length polymorphism (RFLP). This can be used if the presence of the polymorphism in the polynucleotide creates or destroys a restriction site that is recognized by a restriction enzyme. Thus, the treatment of a polynucleotide having this polymorphism will lead to different products produced, compared to the corresponding wild type sequence. Thus, the detection of the presence of specific restriction digestion products can be used to determine the presence of the polymorphism.

The presence of the polymorphism can be determined based on the change that the presence of the polymorphism produces on the mobility of the polynucleotide or protein during a gel electrophoresis. In the case of a polynucleotide, the analysis of single stranded conformation polymorphism (SSCP) can be used. This measures the mobility of the single stranded polynucleotide on a denaturing gel, compared to the corresponding wild-type polynucleotide, indicating the presence of a difference in mobility the presence of the polymorphism. A denaturing gel gradient electrophoresis (DGGE) is a similar system, in which the polynucleotide is electrophoresed through a gel with a denaturing gradient, indicating a difference in mobility, compared to the corresponding wild-type polynucleotide, the presence of polymorphism.

The presence of the polymorphism can be determined using a fluorescent dye and a PCR assay based on an extinguishing agent, such as the TAQMAN ™ PCR detection system. In another method for detecting polymorphism, a polynucleotide comprising the polymorphic region is sequenced through the region containing the polymorphism to determine the presence of the polymorphism.

Various other detection techniques, suitable for use in the present methods, will be apparent to those familiar with the methods of detection, identification and / or distinction of polymorphism. These detection techniques include, but are not limited to direct sequencing, the use of "molecular beacons" (oligonucleotide probes that fluoresce after hybridization, useful in real-time fluorescence PCR; see, for example, Marras et al. , Genet. Anal., 14: 151 (1999)); electrochemical detection (reduction or oxidation of sugars or DNA bases; see US Patent No. 5,871,918 to Thorp et al.); rolling circle amplification (see, for example, Gusev et al., Am. J. Pathol., 159: 63 (2001)); Third Wave Technologies (Madison WI) INVADER® non-PCR based detection method (see, for example, Lieder, Advance for Laboratory Managers, 70 (2000)).

Accordingly, any suitable detection technique as known in the art can be used in the present methods.

As used herein, "determining" the genotype of a subject does not require performing a genotyping technique when the subject has already been genotyped, and the results of the previous genetic test are available; Therefore, determining the genotype of a subject includes referring to previously completed genetic analyzes.

A trial or predictive test kit (patient care) is also described. This assay will help the therapeutic use of pharmaceutical compounds, including NRTI, such as abacavir, based on predetermined associations between genotype and phenotypic response to the therapeutic compound. This essay can take different formats, which include:

(a) an assay that analyzes the DNA or RNA to determine the presence of predetermined alleles and / or polymorphisms. An appropriate assay kit may include one or more of the following reagents or instruments: an enzyme capable of acting on a polynucleotide (generally a polymerase or a restriction enzyme), suitable buffers for enzyme reagents, PCR primers that bind to regions flanking polymorphism, a positive or negative control (or both), and a gel electrophoresis apparatus. The product can use one of the chip technologies described in the art. The test kit shall include printed or readable instructions by means of a machine that offers the correlation between the presence of a specific genotype and the probability that a subject treated with a specific pharmaceutical compound undergoes a hypersensitivity reaction;

(b) an assay that analyzes materials from the body of a subject, such as proteins or metabolites, that indicate the presence of a polymorphism or a predetermined allele. An appropriate assay kit may comprise a molecule, an aptamer, a peptide or an antibody (including an antibody fragment) that specifically binds with a predetermined polymorphic region (or a specific region flanking the polymorphism). The kit may also comprise one or more additional reagents or instruments (as is known in the art). The test kit will also include printed or readable instructions using a machine that offers the correlation between the presence of a specific polymorphism or genotype and the probability of

10 that a subject treated with a specific synthetic nucleoside analog undergo a hypersensitivity reaction;

The biological specimens suitable for the assay are those that comprise cells and DNA and include, but are not limited to blood or blood components, dried blood spots, urine, oral swabs and saliva. Samples suitable for the HLA serological test are well known in the art.

Examples

15 Example 1

Study design

A retrospective case-control study was conducted with adult HIV-infected subjects (> 18 years of age) who participated in a Glaxo Wellcome abacavir clinical development program. The subjects were classified as "case" or "control" subjects based on the following. The case subjects had suffered an episode of suspected or confirmed hypersensitivity to abacavir; Control subjects had received abacavir for at least six weeks, but had not suffered an episode of suspected or confirmed HSR. The six-week treatment period was chosen based on the knowledge that most HSR events occur within the first six weeks of treatment. Case reports were collected at or near the time of the suspected or confirmed hypersensitivity reaction. The control subjects were agreed

25 for the study (if possible) of ethnicity, gender, CD4 + cell count (if available; four intervals of CD4 +: <50, 50-200, 201-500,> 500 cells / mm3); and age (plus or minus 5 years). When possible, the treatment regimen was also agreed ("not exposed to treatment" versus undergoing treatment).

Data collection included demography (age, gender, race, CDC classification for HIV infection);

30 history of food and drug allergies, drug rashes, asthma, eczema, hay fever, etc .; antiretroviral therapy (ART) and concomitant medications taken at the time of HSR (or, for controls, during the first six weeks of treatment with abacavir).

Case-control status, demography and allergy history are provided in Tables 1, 2 and 3.

Table 1- Case-control states (N = 123)

Case-control status

number of concordant No. of subjects

1 case - 2 controls

16 48 (39%)

1 case - 1 control

14 28 (23%)

1 case - 3 controls

one 4 (3%)

1 case - 0 controls

14 14 (11%)

0 cases - 1 control

fifteen 15 (12%)

0 cases - 2 controls

7 14 (11%)

Table 2 - Demography

Cases (N = 45)

Controls (N = 78)

Age, mean (interval)

42 (29-63) 40 (30-62)

Age (years)

18-35

15 (34%) 27 (35%)

35-54

25 (57%) 49 (63%)

> 54

4 (9%) 2. 3%)

Gender

Man

40 (89%) 70 (90%)

Woman

5 (11%) 8 (10%)

Table 3 - History of allergies

Cases (N = 45)

Controls (N = 78)

Any allergy

33 (73%) 46 (59%)

Allergy to sulfa-drugs

14 (31%) 14 (18%)

Any allergy to NNRTI *

9 (20)% Four. Five%)

History of drug rashes

 others 13 (29%) 14 (18%)

Polymorphisms in a series of candidate genes were studied. Polymorphisms studied in the TNFα gene include G (-237) A. The presence or absence of the HLA-B57 allele was also studied.

Example 2

5 TNFα screening

The presence of TNFα G polymorphism (-237A) can be determined using a fluorescent dye and a PCR assay based on an excision agent, such as the allele discrimination form of the 5 'nuclease assay (Lee and Bloch, Nucleic Acids Research, 21: 3761 (1993)). Briefly, this test uses two specific allele probes marked differently with fluorescent "indicator" dyes at the 5 'ends and with the same agent

10 of excision at the 3 'ends. Normally, the fluorescence of each indicator dye is extinguished by the extinguishing agent when it is present in the same olignucleotide molecule. Allele specific probes are used together with two primers, one of which hybridizes with the 5 'template of the allele specific probes, while the other hybridizes with the 3' template of said probes.

The presence of TNFα G polymorphisms (-237) A was determined by the allelic discrimination method,

15 using the 5'-nuclease assay. Two specific allele probes were used with a different fluorescent dye at the 5 'ends, but with the same extinguishing agent at the 3' ends:

TNFα G (-237) A - G: FAM-CCTGCTCCGATTC (MGB) (SEQ ID NO: 9)

TNFα G (-237) A - A: VIC-CCCTGCTCTGATTC (MGB) (SEQ ID NO: 10)

Both probes had a 3 'phosphate group, so AmpliTaq Gold ™ polymerase thermostable polymerase did not

20 I could add nucleotides. The two allele specific probes were designed using a specialized computer program, as is known in the art, so that certain properties (melting temperature, GC content, position of the polymorphic base, location within the amplicon) were made to match. as far as possible, allowing only complete hybridization to the template DNA when the allele specific polymorphic base is present.

The allele specific probes were used together with two primers, one of which hybridizes with the 5 'template of the two allele specific probes, while the other hybridizes with the 3' template of the two probes.

TNFα G (-237) A direct: ATCAGTCAGTGGCCCAGAAGAC (SEQ ID NO: 11)

TNFα G (-237) Inverse: GGGACACACAAGCATCAAGGATA (SEQ ID NO: 12)

If the allele corresponding to one of the specific probes is present, the specific probe hybridizes

30 preferably with the target sequence derived from the mold. The thermostable polymerase, which extends the primer in the 5 'to 3' direction to the allele specific probe, removes the nucleotides from the specific probe, releasing the fluorescent dye and the extinguishing agent. This produces an increase in fluorescence from the indicator dye, which is no longer near the extinguishing agent.

If the allele-specific probe hybridizes to the other allele, incorrect pairing at the polymorphic site inhibits the 5 'to 3' exonuclease activity of the thermostable polymerase and thus prevents the release of the fluorescent indicator dye.

At the end of the thermal cycles of the PCR, the ABI PRISM ™ 7700 sequence detection system is used to measure the increase in fluorescence from each specific dye directly in PCR reaction vessels. Then the reaction information is analyzed. If an individual is homozygous for a specific allele, the corresponding fluorescence is released only to the dye of this specific probe, but if the individual is heterozygous, then the fluorescence of both dyes increases.

The results of TNFα G polymorphism screening (-237) are shown in Table 4.

Table 4

Genotype

Cases (N = 44) Controls (N = 76) p value

1.1 (G / G)

22 (50%) 71 (93%) <0.0001

1.2 (G / A)

20 (45%) Four. Five%) <0.0001

2.2 (A / C)

2 (5%) eleven%) 0.1573

The distribution of TNFα G (-237) A according to various ethnic groups is shown in Table 5 (based on the genetic screening of a genetic population available in the market).

10 Table 5

G / G (1,1) N

A / G (1,2) N A / A (2.2) N Allelic frequency for G Allelic frequency for A

Caucasian (N = 88)

83  4 one 96.6% 3.4%

African Americans (N = 86)

73  13 0 92.44% 7.56%

Hispanic (N = 50)

Four. Five  5 0 95.0% 5.0%

Asians (N = 30)

28  2 0 96.7% 3.3%

Native Americans of the SO (N = 8)

5 3 0 81.24% 18.75%

In the previous study, in the majority of cases the presence of an "A" allele (A / A or A / G genotype) is observed, compared to controls. The G / G genotype appears less frequently in cases, compared to controls.

Example 3

15 Screening of HLA-B57

Genotyping of the HLA-B gene was performed on samples of 120 subjects (44 cases and 76 controls) in the research laboratories of the Anthony Nolan Bone Marrow Trust (Royal Free Hospital, London, United Kingdom). The typing was mainly done using the reference chain-mediated conformation analysis ("Reference Strand-mediated Conformation Analysis" (RSCA); see, for example, Pel-Freez® Clinical Systems,

20 LLC), as is known in the art (Arguello et al., Reviews in Immunogenetics, 1: 209 (1999); Arguello et al., Tissue Antigens, 52:57 (1998)). Sequence-specific DNA and oligonucleotide probes (SSOP) sequencing techniques were used (see, for example, Yoshida et al., Hum. Immunol., 34: 257 (1992); Smith et al., Hum. Immunol., 55:74 (1997)) when necessary, as backup techniques to determine the genotype of HLA.

25 Of all cases, it was discovered that 25/44 (57%) had the HLA-B57 allele (that is, they were heterozygous or homozygous for the HLA-B57 allele), while only 3/76 (4%) of the controls had the HLA-B57 allele (each was heterozygous for the HLA-B57 allele).

Table 6 - Allelic frequency - HLA-B57

Cases (N = 44)

Controls (N = 76) p value

HLA-B57 present

25 (57%) 3. 4%) <0.0001

In subjects who have at least one HLA-B57 allele (cases (n = 25) and controls (n = 3)), the subtype of HLA-B57 is shown in Table 7. The most common allele is B * 5701 ( 24/25 or 96% of the cases carried at least one copy, as well as 1/3 or 33.3% of the controls).

In the present study, the HLA-B57 genotype was found more frequently in cases than in controls.

Table 7 - HLA-B genotype of cases and controls that had at least one HLA-B57 allele

HLA-B genotype

No. of subjects Percentage

CONTROLS N = 3

B * 0801, B * 5701

 one 33.3%

B * 4501, B * 57031

 one 33.3%

B * 4801, B * 57031

 one 33.3%

CASES N = 25

B * 0702, B * 5701

 4 16.0%

B * 07021, B * 5701

 one 4.0%

B * 1402, B * 5701

 one 4.0%

B * 1801, B * 5701

 one 4.0%

B * 35011, B * 5701

 one 4.0%

B * 3503, B * 5701

 one 4.0%

B * 3701, B * 5701

 one 4.0%

B * 3801, B * 5701

 3 12.0%

B * 4001, B * 5701

 one 4.0%

B * 4102, B * 5701

one 4.0%

B * 4402, B * 5701

 one 4.0%

B * 44021, B * 5701

 one 4.0%

B * 4403, B * 5701

 one 4.0%

B * 44031, B * 5701

 one 4.0%

B * 44031, B * 5704

 one 4.0%

B * 4901, B * 5701

 one 4.0%

B * 5501, B * 5701

 one 4.0%

B * 5701, B * 5701

 2 8.0%

B * 5701, B * 57031

 one 4.0%

Example 4

Data were obtained from subjects in addition to those indicated in the previous examples. The other subjects of the retrospective case-control study described in Example 1 were selected for the presence of TNFα 10 G polymorphism (-237) A and HLA-B57. The accumulated data (combining the results provided in the previous examples and the additional data) are indicated in Tables 8 and 9. The total number of subjects was 161; in five subjects there was no

data available for the state of TNFα G (-237) A, and there were no data available in three subjects for the state of HLA B57.

Table 8 - TNFα G (-237) A

Genotype

Cases (N = 57) Controls (N = 99) p value

1.1 (G / G)

32 (56%) 92 (93%)

1.2 (G / A)

23 (40%) 6 (6%) <0.0001 *

2.2 (A / C)

2 (4%) eleven%)

* The P value from the Mantel Haenszel chi-square test indicates a statistically significant difference between the rate of the A allele present in cases versus controls.

Table 9 - Allelic frequency - HLA-B57

Cases (N = 59)

Controls (N = 99) p value

HLA-B57 present

30 (51%) 3 (3%) <0.0001 *

HLA-B5701 present

28 (47%) eleven%) <0.0001

* The P value from the Mantel Haenszel chi-square test indicates a statistically significant difference between the rate of HLA B57 present in cases versus controls.

5 Subjects who have at least one HLA-B57 allele (cases = 30, and controls = 3) were tested to determine the appearance of the HLA B * 5701 subtype. In the cases, 28/30 (93%) had at least one B * 5701 allele; in the controls, 1/3 (33%) had at least one B * 5701 allele.

In the present study, the HLA-B57 genotype was found more frequently in cases than in controls.

Example 5

10 Study design

Additional data from the retrospective case-control study described in Example 1, which includes information from other subjects and information related to other candidate genes, were analyzed. Examples 5 and 6 are accumulated and include the results provided in the previous examples, as well as additional data. The subjects of the previous examples are part of the larger population indicated in examples 5 and 6.

15 A concordant, retrospective and multicenter control case-control study was conducted to identify variants of candidate genes associated with abacavir hypersensitivity. Subjects were adult individuals (≥ 18 years of age) infected with HIV who participated in a GlaxoWellcome abacavir clinical development program (now GlaxoSmithKline (GSK)). Informed consent was obtained. The subjects were classified as "case" or "control." The following criteria were used to identify cases:

20 1. Subjects who suffered symptoms consistent with abacavir hypersensitivity (see # 2 below); these symptoms returned 12 hours after abacavir reexposure; Abacavir treatment was permanently stopped.

2. Subjects who suffered two or more of the following symptoms in a 2-day interval: fever, rash,

Gastrointestinal symptoms (which include nausea, vomiting, diarrhea, abdominal pain) and that permanently stopped treatment with abacavir.

3. Subjects who were diagnosed by a non-GSK doctor who had developed "HSR", "allergic reaction", or "anaphylaxis" attributed to abacavir and who permanently stopped treatment with abacavir.

Before screening a subject as a "case," the diagnosis of abacavir hypersensitivity was evaluated by a GSK physician to determine consistency with the clinical presentation of hypersensitivity.

Concordant controls: HIV-infected adult subjects (18 years of age) who participated in the GSK abacavir clinical development program and who tolerated abacavir for at least 6 weeks with no signs of a hypersensitivity reaction. The control subjects were agreed with a specific case subject based on five criteria when possible: age (more or less 5 years), gender, ethnicity, CD4 + cell count, and regimen of

35 treatment When possible, two concordant controls were recruited for each case enrolled in the study.

Sample management and processing

Blood samples were collected in appropriate blood collection tubes. DNA extraction was performed by DNA Sciences (Morrisville, NC), and the extracted DNA was sent to GSK for genotyping. With the exception of HLA genotyping, genetic tests were performed by GSK. HLA typing

5 was performed by the Anthony Nolan Bone Marrow Trust, London, United Kingdom. The HLA (A, B, and DR) loci were genotyped by the method of reverse chain conformational analysis (RSCA), using DNA sequencing and hybridization of sequence-specific oligonucleotides (SSO) as support (see example 3). Polymorphic markers other than the HLA loci were genotyped using the allelic discrimination form of the 5 'nuclease assay (Applied Biosystems, Foster City, CA; see example 2).

10 Samples were analyzed for the presence or absence of 114 polymorphic alleles.

Subjects of study

A total of 229 subjects were enrolled and provided informed consent. 29 subjects were excluded (the samples produced an inappropriate DNA and / or the subject retrospectively did not meet the inclusion criteria). Two hundred subjects (85 of 100 cases, and 115 of 129 controls) had evaluable data from at least one of 114

15 genetic markers. A total of 157 samples of the subjects were evaluable for TNFα -237, and a total of 197 samples of the subjects were evaluable for HLA-B.

The study population had a median age of 39.8 (24-65) years, and were predominantly male (92%) and Caucasian (74%). Demographic and baseline characteristics were similar between cases and concordant controls. Twenty-seven subjects (14%) were black, and 21 (11%) were Hispanic (table 10).

20 Table 10

Summary of demographic and baseline characteristics according to case-control status

Characteristic

Cases N = 85 Controls N = 115 Total N = 200

Edada (years)

N

 85 115 200

Medium (interval)

40.3 (29-63) 39.8 (24-65) 39.8 (24-65)

18-35 years, n (%)

24 (28) 32 (28) 56 (28)

36-54 years, n (%)

55 (65) 78 (68) 133 (67)

≥55 years, n (%)

6 (7) 5 (4) 11 (6)

Sex

N

 85 115 200

Men, n (%)

79 (93) 105 (91) 184 (92)

Women, n (%)

6 (7) 10 (9) 16 (8)

Ethnicity

N

 85 115 200

Whites, n (%)

66 (78) 82 (71) 148 (74)

Blacks, n (%)

9 (11) 18 (16) 27 (14)

Asians, n (%)

0 0 0

7 (8)

14 (12) 21 (11)

Hispanic Americans, n (%) Others, n (%)

3. 4) 1 (<1) 4 (2)

CDCa class N

 85 114 199

A, n (%) B, n (%) C, n (%) Other, n (%)

31 (36) 16 (19) 36 (42) 2 (2) 43 (38) 22 (19) 45 (39) 4 (4) 74 (37) 38 (19) 81 (41) 6 (3)

a At the time of starting with abacavir

Fifty of the 85 cases (59%) agreed with at least one control, and 41% of the cases had no concordant control (table 11). Reasons why they lack controls include: inability to identify a matching control, and insufficient data. For the 50 cases and their 80 concordant controls, 81% of the controls agreed with a case by age plus or minus 5 years, 99% by gender, 90% by ethnicity, 4% by CD4 cell counts (mainly due insufficient data), and 94% due to the treatment regimen (or participation in the abacavir expanded access program).

Table 11

Summary of the case-control status

HSR-control case status

No. of concordant groups No. of subjects N = 200 n (%)

1 case HSR - 2 controls 1 case HSR - 1 control 1 case HSR - 3 controls 1 case HSR - 0 controls 0 cases HSR - 1 control 0 cases HSR - 2 controls

28 21 1 35 17 9 84 (42.0) 42 (21.0) 4 (2.0) 35 (17.5) 17 (8.5) 18 (9.0)

Example 6

Results

10 Univariate analysis of the genetic association with hypersensitivity

For each polymorphism, allelic frequencies between cases and controls were calculated using univariate analyzes. Polymorphisms (other than HLA polymorphisms) with significantly different frequencies between cases and controls (p value of 0.05 or less) are identified in Table 12 (Fisher's exact test or conditional logistic regression analysis of the difference between rates). The polymorphism of TNF G (-237) A was

15 present in 25 of 58 cases (43%), compared with 7 of 99 (7%) of the controls.

Table 12

Gen (SNP position)

Reference (NCBI dbSNP or GenBank) Variant Cases Controls p value

TNFα (-237)

RS361525 A2 = A 25 (43%) 7 (7%) <0.0001

TNFα (-308)

RS1800629 A2 = A 5 (8%) 28 (28%) 0.0024

TNFα (-5,100)

A2 = G 8 (13%) 30 (31%) 0.0127

MICA (-9,263)

RS1052416 A1 = A A2 = G 48 (92%) 19 (37%) 64 (70%) 66 (72%) 0.0015 <, 0001

MICA (exon 2)

RS1063630 A1 = T A2 = G 40 (83%) 38 (67%) 68 (96%) 47 (48%) 0.0391c 0.0297

MICA (exon 3)

RS1051792 A1 = G A2 = A 46 (77%) 49 (82%) 88 (90%) 57 (58%) 0.0384 0.0029

MICB (exon 2)

RS1065075 A1 = G A2 = A 23 (38%) 48 (100%) 56 (57%) 67 (92%) 0.0334 0.0423c

MICB (exon 3)

RS1051788 A1 = A A2 = G 23 (38%) 48 (100%) 56 (58%) 65 (93%) 0.0209 0.0858c

RNA p47-dependent helicase

A2 = T 27 (45%) 63 (66%) 0.0130

Gen (SNP position)

Reference (NCBI dbSNP or GenBank) Variant Cases Controls p value

ATP

P47-dependent ATP-dependent helicase RNA

RS929138 A1 = C A2 = T 39 (68%) 46 (81%) 37 (39%) 91 (96%) 0.0007 0.0040

ADH7 alcohol dehydrogenase (ADH7-C94T)

Nucleotide 403 in GenBank entry M16286 (5'UTR) A1 = C A2 = T 3 (5%) 45 (96%) 16 (17%) 59 (84%) 0.0431 0.0606c

UDP-glucuronosyltransferase (UGT1A6-A551C)

Nucleotide 765 in GenBank M84130 A1 = A 46 (77%) 59 (60%) 0.0377

a Unless stated otherwise, the p-value is based on Fisher's exact test. b A1 = allele 1, A2 = allele 2. c The value of p is based on a conditional logistic regression between the cases and their concordant controls.

The univariate analysis of HLA typing shows six loci with significantly different frequencies in cases and controls (p <0.1, Fisher's exact test, table 13). Of these, the difference in the frequency of HLA-B57 was the most significant (p <0.0001). HLA-B57 was present in 39 of 84 (46%) cases, compared to 4 of 113 (4%) controls.

Table 13

Summary of HLA alleles through case-control status for p values <0.1a

HLA allele

Cases N (%) N controls (%) p value

HLA-A31

 0 6 (6%) 0.0839

HLA-B08

Four. Five%) 15 (13%) 0.0526

HLA-B57

39 (46%) 4 (4%) <0001

HLA-DRB01

6 (10%) 21 (21%) 0.0826

HLA-DRB03

2. 3%) 18 (18%) 0.0060

HLA-DRB07

23 (38%) 21 (21%) 0.0277

a Fisher's exact test.

Among the non-HLA polymorphisms, the two polymorphisms with the greatest statistical significance were TNFα (237) and MICA (-9263). The HLA-B, MICA and TNFα genes are co-located very close to each other on chromosome 6, and the genotyping results were consistent with the high allelic association between HLA-B57 and the TNFα G allele (-237) A.

10 HLA-B57 and TNFα are co-located very close to each other on chromosome 6, and show a high allelic association (Table 14). In all cases, except two, the hypersensitivity reactions with the TNFα -238A allele were also positive for HLA-B57; five cases of hypersensitivity that were positive for HLA-B57 lacked the TNFα -238A allele.

Table 14

Summary of the association of HLA-B57 and TNFα -237

HLA-B57

TNFα -237 HSR N cases (%) N controls (%)

Subjects without HLA-B57 N

Allele A Without allele A 30 2 (7) 28 (93) 96 5 (5) 91 (95)

Subjects with HLA-B57 present N

Allele A Without allele A 28 23 (82) 5 (18) 3 1 (33) 2 (67)

Multivariate analysis of the genetic association with hypersensitivity

Multivariate exploratory analyzes were performed to investigate the contributions of different markers.

5 genetic Conditional logistic regression was performed using a subset of cases that have at least one concordant control (50 cases and 80 controls, see table 11). A secondary analysis was performed using logistic regression with a total of 85 cases and 115 controls, independent of concordance. These analyzes indicate that HLA-B57 is the most robust marker of the markers studied for hypersensitivity.

The recursive distribution was used to investigate whether combinations of variables, including marker alleles,

10 could be acting to significantly modify the risk of a hypersensitivity reaction to abacavir. HLA-B57 was the most significant predictor of whether a subject was a case or a control (value of p adjusted to Bonferroni <0.0001) (table 15). Of the 159 subjects with sufficient data for inclusion in the recursive distribution analysis, 33 (21%) had HLA-B57; Of these, 30 (91%) were cases. Among the 33 subjects positive for HLA-B57, 31 were negative for DRB03; Within this group of 31 subjects, 30 (97%) were cases (P value adjusted to Bonferroni

15 = 0.001).

Table 15

Summary of the distribution data

or recursive by HLA

Cases HSR N = 84

Controls N = 113

Subjects with ≥1 HLA-B57 allele present, N (%) Subjects without HLA-B57 allele, N (%)

39 (46) 45 (54) 4 (4) 109 (96)

A statistically significant association was found between the presence of HLA-B57 and a history of abacavir hypersensitivity. An association was also discovered between the presence of TNF 237A polymorphism and abacavir hypersensitivity, but this association can be almost completely justified by the presence

20 of HLA-B57. A third polymorphism in the same region, MICA -9263G, was also significant through univariate analysis, but not in multivariate analysis.

Subgroup Analysis

Below are descriptive analyzes of the demographic subgroups. The majority of the subjects in this study were white men. As shown in table 16, 53% of white men cases

25 had at least one HLA-B57 allele, compared to 3% of white male controls. Two of nine cases (22%) of hypersensitivity among black men were HLA-B57 positive, compared with 1 of 16 controls of black men (6%). Among Hispanics and other ethnic groups identified, none of the 9 cases were positive for HLA-B57, compared with 1 of 13 controls.

Table 16

Summary of HLA-B57 data by ethnicity: Men

mens

Ethnicity White Black Other Total

Cases HSR NHLA-B57, n (%)

60 9 9 32 (53) 2 (22) 0 78 34 (44)

Nn controls (%)

74 16 13 2 (3) 1 (6) 1 (8) 103 4 (4)

The majority of the study subjects were men. Of the six cases of women enrolled, five were white (table 17). Four of the five cases of white women were positive for HLA-B57, compared to none of the six controls. Although the association was not statistically tested, the trend corresponds to that of white men.

Table 17 LIST OF SEQUENCES

Summary of HLA-B57 data by ethnicity: Women

Woman

Ethnicity White Black Other Total

Cases HSR NHLA-B57, n (%)

5 0 1 4 (80) 0 1 (100) 6 5 (83)

Nn controls (%)

 6 2 2 0 0 0 10 0

<110> Glaxo Group Limited Seth Hetherington Arlene R. Hughes Eric Lai Michael Mosteller, Jr. Denise Shortino

<120> SELECTION METHOD FOR THE REACTION OF HYPERSENSITIVITY TO A DRUG

<130> PU4539WO 5 <150> 60 / 314.026

<151>

<150> 60 / 336,850

<151>

<160> 13 10 <170> FastSEQ for Windows Version 4.0

<210> 1

<211> 1183

<212> DNA

<213> Homo sapiens 15 <400> 1

<210> 2

<211> 817

<212> DNA 20 <213> Homo sapiens

<400> 2

5

<210> 3 <211> 51 <212> DNA <213> Homo sapiens

<400> 3

cactgggttt gttgcagtaa gccacytcga atgttgctgt agaattaaag t

51

10

<210> 4 <211> 12930 <212> DNA <213> Homo sapiens

<400> 4

<210> 5

<211> 51

<212> DNA

<213> Homo sapiens

<220>

<221> varied configuration

<222> 26

<223> n = A / G

<400> 5

gtgggcagaa gatgtcctgg gagctnagac ctgggacaca gagaccgagg a 51

<210> 6

<211> 51

<212> DNA

<213> Homo sapiens

<220>

<221> varied configuration

<222> 26

<223> n = A / G / C

<400> 6

caggggctcc cggcatttct actacnatgg ggagctcttc 51 ctctcccaaa a

<210> 7

<211> 101

<212> DNA

<213> Homo sapiens

<220>

<221> varied configuration

<222> 51

<223> n = A or T

<400> 7

<210> 8

<211> 51

<212> DNA

<213> Homo sapiens

<220>

<221> varied configuration

<222> 26

<223> n = A / G

<400> 8

ctttggcaat tctatatggt gagctntaaa ggtgggctcc aggtagggat g

<210> 9

<211> 13

<212> DNA

<213> Artificial Sequence

<220>

<223> Homo sapiens

<400> 9

cctgctccga ttc 13

<210> 10 <211> 14 <212> DNA <213> Artificial Sequence

5

<220> <223> Homo sapiens

<400> 10

ccctgctctg attc

14

10

<210> 11 <211> 22 <212> DNA <213> Artificial Sequence

<220> <223> Homo sapiens

fifteen

<400> 11

atcagtcagt ggcccagaag ac

22

twenty

<210> 12 <211> 23 <212> DNA <213> Artificial Sequence

<220> <223> Homo sapiens

<400> 12

gggacacaca agcatcaagg ata

2. 3

25

<210> 13 <211> 720 <212> DNA <213> Homo sapiens

30

<220> <221> varied configuration <222> 452 <223> n = C or G

<400> 13

Claims (7)

1. A method of screening a human subject as an aid to predict the response to the administration of abacavir, which comprises determining whether the subject has an "A" allele at the polymorphic site G (-237) A of TNFα (SEQ ID NO: 1), in which the presence of said TNFα genotype indicates that the subject is at greater risk of a reaction of 5 hypersensitivity to abacavir, compared to the risk of individuals without that allele. 2. A method according to claim 1, further comprising determining whether the subject has one or two copies of an HLA-B57 allele. 3.- A method of screening a human subject as an aid to predict the response to the administration of abacavir, which comprises determining whether the subject has an HLA-B57 allele, in which the presence of said genotype of 10 HLA indicates that the subject is at greater risk of a hypersensitivity reaction to abacavir, compared to the risk of individuals without said allele. 4. A method according to claim 2 or claim 3, wherein said genotype is a genotype that includes one or two copies of the HLA-B * 5701 allele. 5. A method according to any one of claims 2 to 4, wherein said HLA genotype is determined by a method that detects the presence or absence of the HLA allelic DNA sequence. 6. A method according to any of the preceding claims, wherein said subject has not previously been treated with abacavir. 7. A method according to any one of claims 1 to 5, wherein said subject has previously been treated with abacavir. A method according to any of the preceding claims, wherein said subject has been diagnosed with a disorder selected from an HIV infection, HIV disease, AIDS, and AIDS related complex.